Abstract
An expanded GGGGCC hexanucleotide in C9ORF72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It has been proposed that expanded transcripts adopt G-quadruplex (G-Q) structures and associate with proteins, but whether this occurs and contributes to disease is unknown. Here we show first that the protein that predominantly associates with GGGGCC repeat RNA in vitro is the splicing factor hnRNP H, and that this interaction is linked to G-Q formation. We then show that G-Q RNA foci are more abundant in C9 ALS patient fibroblasts and astrocytes compared to those without the expansion, and more frequently colocalize with hnRNP H. Importantly, we demonstrate dysregulated splicing of multiple known hnRNP H-target transcripts in C9 patient brains, which correlates with elevated insoluble hnRNP H/G-Q aggregates. Together, our data implicate C9 expansion-mediated sequestration of hnRNP H as a significant contributor to neurodegeneration in C9 ALS/FTD.
Highlights
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related syndromes that occur alone or together in families, and sporadically in individuals
While previous studies have identified a number of possible C9 RNA binding proteins (Haeusler et al, 2014; Lee et al, 2013; Cooper-Knock et al, 2014; see Discussion), we investigated this in a way that allowed us to analyze direct binding with sensitivity and selectivity, and to determine the possible influence of G-Q formation on binding
We investigated whether the G-Q/hnRNP H foci we detected by immunostaining of patient cells sequester sufficient amounts of hnRNP H to bring about the splicing changes we detected in C9ALS brains
Summary
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related syndromes that occur alone or together in families, and sporadically in individuals. GGGGCC (G4C2) in an intervening sequence separating two putative first, noncoding exons in the gene C9ORF72 (C9) (DeJesus-Hernandez et al, 2011; Renton et al, 2011) is the most frequent known cause of both disorders. How this expansion leads to disease is unclear, several non-mutually exclusive mechanisms have been suggested. In the case of C9ALS, sequestration of RNA-binding proteins by the transcribed G4C2 expansion is a proposed pathogenic mechanism, and it has been shown that RNA from this locus forms intranuclear foci in the brains of affected individuals (Donnelly et al, 2013). It has been shown that poly dipeptide repeat (DPR) proteins are translated from repeat-containing transcripts, and DPRs may contribute toxic effects of their own (Mori et al, 2013b; Kwon et al, 2014; Wen et al, 2014)
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